Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery.

BACKGROUND AND PURPOSE

The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation.

EXPERIMENTAL APPROACH

Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured.

KEY RESULTS

A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA.

CONCLUSIONS AND IMPLICATIONS

Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.